Ricin toxin (RT or ricin) is a naturally occurring toxin composed of an enzymatically active, cytotoxic "A" amino acid sequence, and a "B" sequence, which is presumed to be responsible both for attaching the "A" sequence to a target cell to be killed, and to aid in the translocation of A fragment into the cytoplasm. Other examples of such toxins include diphtheria toxin and the exotoxin from Pseudomonas aeruginosa. Other toxic proteins, such as, for example, those derived from Phytolacca americana (PAPI, PAPII, and PAP-S) and gelonin show in vitro activities comparable to the "A" sequences of the above toxins, but are inactive in vivo, presumably due to the absence of a "B" chain.
The "ricin" peptides of the present invention are derived from the seeds of Ricinus communis, commonly known as castor beans. Two similar proteins (often called lectins) are extractable from these seeds: the above-mentioned ricin and Ricin communis agglutinin (RCA). Both proteins contain A and B portions; however, the A and B portions do not comprise a single peptide. The A portions of these moieties are capable of catalytically inactivating the large subunit of ribosomes in vitro and the mechanism of ricin for in vivo cytotoxicity is believed to reside in this capacity for ribosome inactivation. Ricin and RCA appear to be highly homologous (Cawley, D. B., et al, Arch Biochem Biophys (1978) 190:744) but differences exist. RCA is dramatically less toxic, and appears to exhibit characteristics corresponding to those expected of a dimer of ricin.
The components of ricin and of RCA have been well characterized and sequenced on the basis of the extracted materials. (Funatsu, G., et al, Agric Biol Chem (1979) 43:2221). Ricin has an apparent molecular weight of 58,000 daltons and consists of the A chain with a molecular weight of 32,000 daltons and a B chain of molecular weight of 34,700 daltons. RCA is a tetramer which has two A subunits of molecular weight 32,000, and two B subunits of molecular weight 36,000 each. In their native environments, the B chains are generally glycosylated. The A and B subunits of both ricin and RCA are linked only by a single disulfide bond, and not by peptide linkage (Funatsu, G., et al, Agri Biol Chem (1977) 41:1211) unlike, for example diphtheria toxin which is found as a single chain peptide. It is also known that both ricin and RCA, though having separate peptides for A and B portions, are derived from a single chain precursor in each case (Butterworth, H. E., et al, Eur J Biochem (1983) 137:57). As a result of the work related to the present invention, it has been shown that the single chain precursor appears to contain a sequence of 12 amino acids between the A chain (amino terminal) and B chain sequence. It is assumed that upon excision of the dodecameric intervening peptide, the A and B chains remain linked through the single disulfide bond.
The present invention provides a means for obtaining the B chain of ricin using recombinant technology thus providing with greater accuracy the entire amino acid sequence, and making possible an exploration of the structural features required for its activity. The techniques and materials of the present invention further permit selective modification of the amino acid sequence of the B chain and thus permit manipulation to provide properties which are capable of enhancing the cytoxicity of ricin or of other toxins and the derivatives thereof. By enabling the production of ricin B chain using predictable, efficient, and economic procedures which, further, permit directed modification, the invention permits the use of B chain in practical and improved ways not before possible.